Antiphagin and artiviral system based on DNA sulfur phosphorus acylation modification

A phosphorothioylation and anti-phage technology, applied in the field of anti-phage and anti-virus systems, can solve the problems of S.enterica double-stranded DNA damage, cell structure changes, growth defects, etc.

Active Publication Date: 2019-07-30
WUHAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Further studies have shown that once the phosphorothioation modification is removed, DndFGHI can cause S.enterica double-strand DNA damage, resulting in growth defects, triggering SOS responses and cellular structural changes (Gan, R. et al., 2014.Sci Rep. 4:6642)
Although these features are similar to the "self / non-self recognition" strategy of the methylation-modified R-M system, there are currently no reports suggesting that the dndABCDE-dndFGHI system has the function of resisting phages or viruses

Method used

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  • Antiphagin and artiviral system based on DNA sulfur phosphorus acylation modification
  • Antiphagin and artiviral system based on DNA sulfur phosphorus acylation modification
  • Antiphagin and artiviral system based on DNA sulfur phosphorus acylation modification

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Anti-phage and anti-viral gene clusters based on DNA phosphorothioylation modification.

[0033] There are two types of DNA phosphorothioylation modification. One is the double-stranded phosphorothioylation modification guided by the dnd gene cluster. Taking Salmonella enterica serovar Cerro 87 as an example, there is dndFGHI around the modified gene cluster dndABCDE. Identification of whether dndABCDE-dndFGHI contributes to phage resistance. The other is the single-strand phosphorothioylation modification mediated by the sspABCD gene cluster. Taking Vibrio cyclitrophicus FF75 as an example, a gene with nuclease activity was found downstream of sspABCD, and it was named sspE. We also found a modifier gene cluster of dndCDEA in archaea, and found a homologous gene of the type III restriction subunit in the region adjacent to the modifier gene cluster, which is a gene related to the defense of DNA phosphorothioation modification, and its surrounding The gene w...

Embodiment 2

[0034] Example 2: Construction of plasmid pWHU3930 containing dndBCDEFGHI

[0035] The plasmid pJTU1238 containing dndBCDE was digested by SacI and SpeI, and the linear vector DNA containing SacI and SpeI cohesive ends was recovered, using 5'-AAAGGGAACAAAAGCTGGAGCTCAAAGGTCGGTATTCCGA-3' and 5'-ACATGCACCGAAATAGCATGCTCTTTTTCTTGCTTA-3' as primers, and Salmonella enterica serovarCerro 87 Genomic DNA of Salmonella enterica serovar Cerro 87 was used as a template to obtain a fragment 1 with a length of 5.6 kb by PCR, using 5'-TAAGCAAGAAAAAGAGCATGCTATTTCGGTGCATGT-3' and 5'-AACCGGCCGCAGATCCACTAGTAATAATATGGCACCATG-3' as primers, and using the genomic DNA of Salmonella enterica serovar Cerro 87 as a template, obtained by PCR Fragment 2 with a length of 4.6 kb. By Gibson splicing, fragment 1, fragment 2 and the linear vector DNA digested by SacI and SpeI were ligated to obtain the recombinant plasmid pWHU3930( figure 1 ) containing the dndBCDEFGHI gene cluster from Salmonella enterica se...

Embodiment 3

[0036] Example 3: Plasmid pWHU3930 containing dndBCDEFGHI confers host resistance to phage

[0037] The pWHU3930 was introduced into NEB 10-beta, and NEB 10-beta (pWHU3930) was obtained through ampicillin resistance selection, and pJTU1238 was introduced into NEB 10-beta, and NEB 10-beta (pJTU1238) was obtained through ampicillin resistance selection. NEB 10-beta (pJTU1238) and NEB 10-beta (pWHU3930) were cultured overnight at 28°C. The next day, expand into 5 mL LB medium at a volume of 1:100, and culture at 28 degrees until the OD600 is 0.8. Take 400 μL of the bacterial solution, prepare a plate by the double-layer plate method, dilute the phage step by step, and spot the plate. Invert the plate and place it in a 28°C incubator. The next day, the infection status of phages in NEB 10-beta (pWHU3930) and NEB 10-beta (pJTU1238) was observed. The result is as figure 2 , the bacteria containing the dndBCDEFGHI system have obvious resistance to bacteriophage T7, T4 and their ...

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Abstract

The invention discloses an antiphagin and artiviral system based on DNA sulfur phosphorus acylation modification, and relates to antiphagin systems in bacteria and antiviral systems in archaea. On onehand, in bacteria, type-I sulfur phosphorus acylation modification gene clusters dndABCDE and dndFHGI gene clusters can protect hosts to resist infection of phages, and the antiviral system in archaea is composed of dndCDEA-pbeABCD gene clusters; on the other hand, the invention further discloses type-II antiphage systems composed of sspBCD-sspE, and infection of the phages of the bacteria can also be resisted. The antiphagin and artiviral system can be applied to development of biotechnologies and industrial fermentation technologies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-phage and anti-virus system related to DNA phosphorothioylation modification. Background technique [0002] DNA phosphorothioylation modification is a natural modification on the DNA backbone found in bacteria in recent years. Unlike methylation modification that occurs on DNA bases, DNA phosphorothioylation modification occurs on the DNA backbone. atom replaces a non-overseas oxygen atom on the DNA phosphodiester bond, and the R P Configurations exist (Wang, L. et al., 2007. Nat Chem Biol. 3:709-10; Eckstein, F. et al., 2000. Antisense Nucleic Acid Drug Dev. 10:117-21). Studies have found that there are two types of DNA phosphorothioylation in nature so far, namely type I phosphorothioylation mediated by the dnd gene cluster and type II phosphorothioylation mediated by the ssp gene cluster (Cao , B. et al., 2014. Nat Commun.5:3951). [0003] Type I phosphoroth...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N1/21A01N63/00A01P1/00C12R1/19C12R1/01
CPCA01N63/00C07K14/195C07K14/255C12N15/70C12N15/74
Inventor 陈实李相烨王连荣邓子新刘思怡肖瑶邹璇陈超姜先月李梦雪熊磊
Owner WUHAN UNIV
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